Distinctive chemotactic responses of three marine herbivore protists to DMSP and related compounds

Abstract Marine planktonic predator–prey interactions occur in microscale seascapes, where diffusing chemicals may act either as chemotactic cues that enhance or arrest predation, or as elemental resources that are complementary to prey ingestion. The phytoplankton osmolyte dimethylsulfoniopropionate (DMSP) and its degradation products dimethylsulfide (DMS) and acrylate are pervasive compounds with high chemotactic potential, but there is a longstanding controversy over whether they act as grazing enhancers or deterrents. Here, we investigated the chemotactic responses of three herbivorous dinoflagellates to point-sourced, microscale gradients of dissolved DMSP, DMS, and acrylate. We found no evidence for acrylate being a chemotactic repellent and observed a weak attractor role of DMS. DMSP behaved as a strong chemoattractor whose potential for grazing facilitation through effects on swimming patterns and aggregation depends on the grazer’s feeding mode and ability to incorporate DMSP. Our study reveals that predation models will fail to predict grazing impacts unless they incorporate chemotaxis-driven searching and finding of prey.


Supplementary Figure S2. Cell density distributions in the microcapillary assays depicted by histograms.
Cell density distributions of K. armiger, O. marina and G. dominans in microcapillary assays depicted by histograms.The figure shows the distributions in all replicates, substrates, and concentrations.Note that the capillary with substrate (dashed lines) is always compared with a control without substrate (filled area).The 0 on the X axis of the histograms represents the capillary entrance, with the inside area on the left (negative distance) and the outside on the right (positive distance).Supplementary Figure S3.Cell density temporal evolution.Total cell concentration in the two zones of interest was followed through all the incubation.Cell concentration inside and outside was totally calculated to observe temporal patterns in K. armiger, O. marina, and G. dominans microcapillary assays.The figure shows the total cell concentration in all replicates, substrates, and concentrations.Note that the capillary with substrate (dashed lines) is always compared with a control without substrate (filled area).S1.Linear regression results of K. armiger cell concentrations over time inside the capillaries.Accumulation in the DMSP capillary (cell µm 2 s -1 ) was assessed by subtracting the slope in the control capillary (C) from the slope in the DMSP capillary (D).Std.err: standard error of the slope; p: probability of slope=0; Rep: replicate; err(D-C): propagated error of the slope subtraction.S2.Linear regression results of O. marina cell concentrations over time inside the capillaries.Accumulation in the DMSP capillary (cell µm 2 s -1 ) was assessed by subtracting the slope in the control capillary (C) from the slope in the DMSP capillary (D).Std.err: standard error of the slope; p: probability of slope=0; Rep: replicate; err(D-C): propagated error of the slope subtraction; err: propagated error of the mean.Instantaneous swimming speed.G. dominans showed a clear increase in swimming speed when tested with DMSP 20 µM and 200 µM (Supplementary Fig. S4).The histograms in the upper part of the panel display the observed relative frequency of swimming speeds in each repetition in 3 different zones: near the entrance of the DMSP filled capillary, near the control capillary and in the remaining frame area.Cells swimming close to the DMSP source (r < 200 µm) show a shift towards higher velocities (between 200 µm/s and 300 µm/s) in comparison to the rest of the cells.The colormaps in the lower part of each panel show the frequency of the observed swimming speed as a function of the distance from the DMSP source.A patch of cells swimming between 200 µm•s -1 and 300 µm•s -1 is distinguished in the upper part of the colormap corresponding to shorter radial distances.Contrastingly, cells further away from the source move typically below 150 µm•s -1 .The patch of fast swimming cells extends in most repetitions up to 200 µm from the DMSP source, except for R3 at 20 µM and R2 at 200 µM in which the patch extends up to approximately 400 µm.In most repetitions, we observe also a patch of slowly moving cells (< 70 µm•s -1 ) at the entrance of the capillary.This behaviour is sometimes observed also in the control capillary (R2 at 2 µM, R4 at 20 µM, R2 at 20 µM) and may be caused by cells whose flagella became trapped at the pipette entrance.These cells slowly oscillate around the capillary entrance until they successfully break free and return to the bulk.A less clear chemokinetic behaviour is observed for repetitions at 2 µM.A mild chemokinetic effect is observed in R1, while in repetitions R2 and R3 the cells swim fast (between 200 µm•s -1 and 300 µm•s -1 ) in the entire frame area and not only near the DMSP source as in the other repetitions.Supplementary Table S4

EXTENDED MATERIALS & METHODS
Diffusion experiment from a capillary of squared cross-section.The experimental design required both capillaries in the same frame and thus they were placed in close proximity.Consequently, after some time the control capillary would be reached by the diffusing DMSCs plume from the test capillary.The typical timescale needed for the plume to reach the control capillary can be approximated as: where D is the diffusion coefficient of the solute and x the distance between the two capillaries.In the experiment, the distance between test and control capillaries was typically ~1 .Knowing the diffusion coefficients of DMSP (  "#$% ~6 * 10 &'  ! ⁄ ), DMS (  "#$ ~1.1 * 10 &(  ! ⁄ ) and acrylate ( )*+,-)./~1.1 * 10 &(  ! ⁄ ), the times after which the plumes would reach the entrance of the control capillary were τ "#$% ~ 8  , τ "#$ = τ )*+,-)./~ 4 , i.e., within the duration of most video recordings.Nevertheless, we expected that the presence of a continuous DMSCs source would maintain a much stronger gradient at the entrance of the test capillary for the whole duration of the experiment.To confirm the expected gradients, we recorded the diffusion process from a square-shaped CM Scientific microcapillary immersed in L1 medium for 20 minutes using Allura Red as solute (Supplementary Fig. S5).The strength of the Allura Red gradients during the recording time at the source and 1 mm away (points A and B in Supplementary Fig. 1a) was quantified by image analysis.
The diffusion constant of Allura Red (  0--1+) 3/4 ~5 * 10 &'  ! ⁄ ) is almost the same as that of DMSP and half those of DMS and acrylate.Accordingly, the Allura Red gradients measured 20 minutes after the start of the diffusion process will thus be comparable with those of DMSP after the same time and those of DMS and acrylate after 10 minutes.Gradients were calculated for a scalar field S ranging between 0 and 1 proportional to a color linear scale (0 = black pixel, no Allura Red, 1= gray pixel, max Allura Red concentration inside the capillary) in units of µm -1 .Gradients are calculated radially over a semicircle centered at the entrance of the capillary θ ∈[-π/2, π/2] (point A), where θ =0 corresponds to the direction parallel to the capillary.The same was done at point B one millimeter away from A. The temporal evolution of the gradients is shown in the left panel of Supplementary Fig. 1b.The histograms (right panel Supplementary Fig. 1b) represent the discrete probability distribution of the gradients dS/dr integrated for all values of θ ∈[-π/2, π/2] over the total recording time (sampled once every minute).The results confirm that strong gradients were maintained near the DMSCs source compared to the control capillary for all compounds (DMSP, DMS, acrylate) for the whole duration of a 10-min.experiment.
Figure S4.Probability distribution of the instantaneous swimming speed of G. dominans at each DMSP concentration.Each panel contains two plots.The top chart compares the relative frequencies of the observed swimming speed within 3 different zones, represented in 3 colours: (i) orange: r < 200 µm from the entrance of the DMSP-filled capillary, (ii) green: r < 200 µm from the entrance of the control capillary and (iii) grey: the remaining frame area (r > 200 µm from both capillaries).The bottom chart is a colormap of the relative frequency of the instantaneous swimming speed at different radial distances from the entrance of the DMSP-filled capillary.

Supplementary Table S3. Linear regression results of G. dominans cell concentrations over time inside the capillaries.
Accumulation in the DMSP capillary (cell µm 2 s -1 ) was assessed by subtracting the slope in the control capillary (C) from the slope in the DMSP capillary (D).Std.err: standard error of the slope; p: probability of slope=0; Rep: replicate; err(D-C): propagated error of the slope subtraction; err: propagated error of the mean.

. Median values, errors, and significant differences of cellular densities (cell/µm 2 ) from both capillaries (Cap. C=control and S=substrate-filled) in K. armiger incubations.
Significant differences were evaluated with the nonparametric Kruskal-Wallis test.The statistical test was applied to each concentration (2-200µM), and cue (D=DMSP, M=DMS, A=ACRYLATE, Std.D=standard deviation, N=number of frames, Std.E= standard error).